1. Field of the Invention
The present invention generally relates to an automated method for detecting circulating cancer cells in an individual. There is presented in one embodiment a method of assessing treatment protocols in the treatment of one or more cancers.
2. Description of the Related Art
Primary tumor cells presumably spread to other organs via blood and lymphatic circulation (1), and are probably shed from tumors at all stages, often well before metastasis. Circulating tumor cells (CTC) are likely to reflect both tumor cell bulk and tumor growth (2, 3).
Identification and counting CTC when they are very rare (at most a few CTC per ml) could be an indication of a cancer, or even of a precancerous growth before the appearance of evident clinical symptoms. The potential interest in the detection of CTC in peripheral blood was first suggested over a century ago (4) but because of their very low numbers CTC cannot be detected by conventional methods. The challenge is to develop an approach that combines high sensitivity with high specificity for the identification and characterization of rare tumor cells circulating in the blood enabling them to be distinguished from normal epithelial cells and leukocytes.
Clear-cut detection of circulating tumor cells could facilitate cancer prognosis, diagnosis of minimal residual disease, assessment of tumor sensitivity to anticancer drugs, and personalization of anticancer therapy. A highly sensitive and specific identification of CTC could also have a potential application in the early diagnosis and screening of invasive cancers.
Tumor cells, epithelial cells, platelets and mononuclear white cells have been enriched from whole blood, and specifically separated from neutrophils and erythrocytes, by a variety of approaches including immunomagnetic separation, density gradients and filtration (10-13). The separated cells can then be collected for cytopathological analyses, immunolabelling and molecular studies. Various methods have been used for the detection and enumeration of CTC. These include flow cytometry, nucleic acid based approaches (RT-PCR, methylation specific PCR) and immunofluorescence microscopy (4-9).
RT-PCR based methods analyze the expression of genes specific to epithelial cells or to the normal tissues from which the tumor cells originate. The main potential advantage of this approach is its high sensitivity. However, since cell integrity is destroyed during RNA extraction, this approach precludes the analysis of cell morphology and phenotype and so, in particular, may not be able to distinguish material shed directly from normal tissue as opposed to from tumors nor does it allow the detection of several associated changes in the same cell. RT-PCR tests thus do not enable counting of CTC and can, in general, only give a positive or negative response that depends on the sensitivity of the test and on the amount of blood that can be tested. In addition, RT-PCR tests cannot distinguish between circulating normal and cancer cells since they nearly always detect tissue-specific and not cancer-specific mRNAs in peripheral blood (14, 15).
Immunofluorescence microscopy enables analysis of cell morphology and direct counting of identifiable presumptive tumor cells. Detection is carried out by immunolabeling of cells using appropriate antibodies. However, since there are so far no antibodies for tumor specific antigens, antibodies specific to epithelial antigens have been used to identify CTC, for example, EpCAM, BerEP4, and epithelial specific Cytokeratins (16-19). Other markers that are not necessarily specific to epithelial cells have also been used. These include antibodies to mammaglobin, PSA, CEA, surviving and HER-2 (8, 20). However, false negative results can occur since these antigens are not present in all tumor cells. Despite these difficulties, the literature in general shows the presence of increasing numbers of CTC in patients having more advanced cancers, higher risk of relapse and poor prognosis (4, 21-23).
There is a need therefore for a highly sensitive, reliable, and quantitative detection method for CTC in the blood that overcomes the short comings of prior methods.